Föreläsning 9: Plant biotechnology II

What is mutation breeding

Creating new plant varieties by inducing mutations using radiation or chemicals

Describe radiation induced mutagenesis

• Flourished in 1940s and 50s

• Radiation can induce changes in basepairs but also changed due to DNA breakage → insertions/deletions and translocation

What is the most commonly used chemical in chemically induced mutagenesis

• EMS (Ethyl Methanesulfate)

• Alkylates guanine in 6th position = break one of the hydrogen bonds to cytosine

• The modified G can bind to thymine → replication changes where G-C pairs to A-T, causing point mutation (SNP)

• Must titrate EMS carefully because too much kills seeds and too little causes few mutations

What are the possibilities with a mutation population

• Increases variation

• You don’t need to know what gene you are looking for

• Give new traits

• Can give info about different genes

• Easy to compare because genetic background is the same

• Unnecessary mutations can to large extent be removed by backcrossing

RNA interference (RNAi) is a method for gene knockdown. Describe it

• A a post-transcriptional gene silencing technique to reduce (not completely stop) the expression of a gene.

• dsRNA targets mRNA with the same sequence for breakdown

• Not 100% stop of translation, there will be some “leakage”

• Applications:

  • delay tomato ripening (by targeting polygalacturonase).

  • Can also target pests or viruses (e.g., dsRNA added to plant surface, does not need to be produced by the plant).

There are two ways to introduce dsRNA in RNAi. Describe them

Antisense RNA:

• When cloning, introduce a gene in reverse orientation to the target gene, by a promotor

• Transcription gives an antisense (complementary to the mRNA)

• Antisense and mRNA binds → dsRNA

• The cells’ nuclease target the dsRNA and degrades it

Double-stranded RNA:

• Introduce an antisense next to the the target gene

• In transcription both the sense and antisense are transcribe to mRNA

• The antisense binds to the sense which forms a hairpin RNA (double-stranded

• The cells’ nuclease target the dsRNA and degrades it

Compare gene knockdown to gene knockout (e.g CRISPR)

• Differences

  • Knockdown:

    • not completely knocked out = some leakage

    • controlled by promotor → can therefore be tissue specific

  • Knockout:

    • completely knocked out → no protein produced

    • in all cells and tissues

• Similarities

  • Several homeologs can an be simultaneously targeted

  • Knockdown: New DNA added → GMO

  • Knockout: depending on approach, DNA can be added or not → currently considered GMO

Why is it easier to modify potatoes through gene technology than traditional breeding

• Potatoes are very difficult to cross and breed:

  • genome is tetraploid and very heterozygote

  • crossing to wild relatives is not possible

• Therefore easier with gene technology

Describe the approach to modify potatoes through CRISPR-Cas9

• Potatoes have two types of starch:

  • amylopectin (branched)

  • amylose (straight)

• A single gene, GBSS, codes for the protein needed to produce amylose

• When GBSS is knocked out, only amylopectin is obtained

Steps:

1. Create protoplasts (plant cells without cell walls).

2. Introduce CRISPR/Cas9 system that knocks out the GBSS gene by making a cut that gets improperly fixed - causing a change that knockouts the gene.

3. Regenerate to callus → whole plants from edited cells.

4. Screen for desired genetic changes.

Where do new genes come from in nature

• Whole genome duplication

• Tandem duplication (gene copies side by side)

• TE-mediated insertions

• Horizontal gene transfer (from other species)

• De novo gene birth

• Introgression

• Segmental duplication

• Biotech can mimic these natural processes to introduce new traits.

Name the different ways to knockout a plant gene

• Crossings with a relative with mutated/inactive gene – Changes half
  the genome at first crossing (can be backcrossed, about 1% of
  genome changed after 6 backcrosses)

• Mutagenesis – induces mutations throughout the genome (can be
  backcrossed)

• T-DNA insertion –  Uses Agrobacterium to insert DNA randomly, often disrupting genes.

• CRISPR for homologous recombination –  Guides CRISPR to a specific site and swaps DNA via a template - Precise gene knockout.

• CRISPR (or ZNF/TALEN) to introduce NHEJ event – Creates small deletions/insertions by cutting DNA and letting the cell "repair" it.

• CRISPR base editing – a single base edited

What are the 5 most common GM plants today (the big 5)

1. Soybean

2. Maize

3. Cotton

4. Canola

5. Alfalfa

In which global areas are GM plants produced and used

• GMOs are approved in 41 countries and grown in 22.

• North and South America dominate usage.

• Europe is more restrictive (e.g., only Spain grows GM maize).

• Crops grown include maize, cotton, soybean, papaya, and more.

What are the GM crops engineered for

• Insect resistance

• Herbicide resistance

• Abiotic stress tolerance

• Disease resistance

• Nutritional improvement

What are the concerns against transgenic crops

1. Biosafety

2. Resistance breakdown

3. Adverse effects on non-target organisms

4. Cost for commercialization

5. Oligopoly of multinational companies

Why is biosafety a concern and what are the answer to them

Concerns:

• The issue of potential health risk of toxicity and allergenicity

• Transfer of antibiotic resistance (used as marker genes)

• Adverse effects on the environment

Answers:

• Overwhelming amount of studies show no adverse health effects

  • But must be evaluated in each case

• Likelihood of transfer of antibiotic resistance extremely low

  • Other selection systems

  • Marker free methods

• Herbicide resistant weeds, uncommon, but has occurred. Crucial to have a strategy to minimize this risk

  • Editing of chloroplast genome – no transgenic pollen

  • Chloroplast transformation more difficult – but results in higher amount of protein
    produced

What are the strategies against resistance breakdown

• Production of multiple toxins

• Rotate crops or toxin

• Plant non-GM “refuge” zones to slow resistance

What is the EU GMO definition

“organisms in which the genetic material (DNA) has been altered in a way that does
not occur naturally by mating or natural recombination”

What methods used for gene knockout are not considered as GMO

• Crossings with a relative with mutated/inactive gene

• Mutagenesis: GMO by definition but not regulated as GMO by regulation

What methods used for gene knockout are considered as GMO

• T-DNA insertion: also overexpression and RANi

• CRISPR for homologous recombination

• CRISPR (or ZNF/TALEN) to introduce NHEJ

• CRISPR base editing

Natural mutagenesis, induced mutagenesis and CRISPR can all result to the same SNP but currently differently regulated in EU

What is the purpose of the EU regulation regarding GMO

“The GMO(CU) Regulations provide for human health and safety and environmental protection
from genetically modified micro-organisms (GMMs) in contained use, and human health and safety from genetically modified plants and animals.”

Describe the two step authorization of GMO in EU

1. Scientific Risk Assessment
   👨‍🔬 Done by EFSA. Thorough but slow and expensive.

2. Political Risk Management
   🇪🇺 EU Commission and member states decide if it can be approved.
   - Often delayed due to political disagreement.

Also includes:

• Labeling (costly)– for consumer transparency.

• Co-existence rules with organic and conventional crops and costly

What are the new regulations proposed for NGT (new genomic techniqes)

• EU is proposing a new system for regulating gene-edited plants:

  • NGT1 category: Considered equivalent to conventional plants if:

    • Max 20 edits

    • No foreign DNA

    • Cannot be herbicide tolerant

    • Cannot be used in organic farming

NGT2 category: Treated as GMO, needs full authorization.

Describe mapping-by-sequencing

What mutation is responsible for the phenotype in EMS (mutagenized) population?

1. Backcrossing mutant with wild-type: späder ut alla irrelevanta mutationer

2. Self-fertilization of F1 to get segregation in F2/F3: Vissa får mutationen, andra inte. (F1 = generation 1, första avkomman)

3. Pool plants based on phenotype: e.g., “BB” (mutant) vs “bb” (wild-type).

4. Sequence both pools → identify mutations present only in the mutant pool.